The sequencing of the human genome has created the promise and opportunity for understanding the function of all genes and proteins relevant to human biology and disease, Peltonen and McKusick, Science, 291: 1224-1229 (2001). However, several important hurdles must be overcome before this promise can be fully attained. First, even with the human genome sequence available, it is still difficult to distinguish genes and the sequences that control their expression. Second, although monitoring gene expression at the transcript level has become more robust with the development of microarray technology, a great deal of variability and control of function originates in post-transcriptional events, such as alternative splicing and post-translational processing and modification. Finally, because of the scale of human molecular biology, potentially many tens of thousands of genes, and their expression products, will have to be isolated and tested in order to understand their role in health and disease, Dawson and Kent, Annu.Rev.Biochem., 69: 923-960 (2000).
In regard to the issue of scale, the application of conventional recombinant methodologies for cloning, expressing, recovering, and isolating proteins is still a time consuming and labor-intensive process, so that its application in screening large numbers of different gene products for determining function has been limited. Recently, a synthesis approach has been developed which can address the need for facile access to highly purified research-scale amounts of protein for functional screening, Dawson and Kent (cited above) and Dawson et al., Science, 266: 776-779 (1994). In its most attractive implementation, an unprotected oligopeptide intermediate having a C-terminal thioester reacts with an N-terminal cysteine of another oligopeptide intermediate under mild aqueous conditions to form a thioester linkage which spontaneously rearranges to a natural peptide linkage, Kent et al., U.S. Pat. No. 6,184,344. The approach has been used to assemble oligopeptides into active proteins both in solution phase, e.g. Kent et al., U.S. Pat. No. 6,184,344, and on a solid phase support, e.g. Canne et al., J. Am. Chem. Soc., 121: 8720-8727 (1999). Recently, the technique has been extended to permit coupling of C-terminal thioester fragments to a wider range of N-terminal amino acids of co-reactant peptides by using a removable ethylthio moiety attached to the N-terminal nitrogen of the co-reactant, thereby mimicking the function of an N-terminal cysteine, Low et al., Proc. Natl. Acad. Sci., 98: 6554-6559 (2001).
Presently, the synthesis of oligopeptide thioesters is carried out primarily using t-butoxycarbonyl (“Boc”)-based protecting groups. Alternatively, 9-fluorenylmethoxycarbonyl (“Fmoc”)-based protecting groups can also be used, but only when the reaction times are short, as the nucleophilic reagants used to remove the Fmoc protecting groups also degrade the thioesters of the oligopeptides over time, as noted by Botti et al., International patent publication WO 02/18417. To address this problem, Botti et al. have suggested the use of a nucleophile-stable thioester precursor that is removable under non-nucleophilic conditions, in combination with a nucleophilic labile Fmoc protecting group as the N-terminal protecting group. While the work of Botti et al. does facilitate the coupling of amino acids and oligopeptides, the 2-mercapto carboxyesters of Botti et al. are susceptible to nucleophilic cleavage during long reaction times. As the preparation of very long oligopeptides, polypeptides and proteins requires extended reaction times, a new method that provides stable N-terminal and C-terminal protecting groups for the preparation of oligopeptides, polypeptides and proteins, would be advantageous. Surprisingly, the present invention provides such methods.